Chapter 4 and 5 Ig study questions: How does the immune system recognize diverse possible antigens? How do antibodies simultaneously recognize a huge variety of antigens and carry out a limited number of effector functions? What is V(D)J recombination? What do the letters (and parentheses) signify? What is the 12/23 rule? What are RAG-1/2 and TdT? What do they recognize/do? What molecular changes are required to switch from membrane-bound to secreted Ig? I will be mixing the two chapters, which I believe is the more logical form of presentation. But reading through sequentially also has merits. For complete understanding, you read the textbook chapters, attend the lecture (taking notes obviously), and study this lecture file. I will not be covering the T cell portions of the two chapters. Those sections will be covered in Dr. Evavold’s and Williams’ lectures.

Ig Structure and Function Lecture – Sept. 5, 2017 Dean Tantin, PhD, Department of Pathology Division of Microbiology & Immunology University of Utah School of Medicine dean.tantin@path.utah.edu

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Abcam (an Ab company) (B cell receptor, BCR) For any antibody, in a natural situation, the heavy chain polypeptides are identical to each other. Similarly, the light chain polypeptides are identical to each other. This is because they are each the products of a single gene: on of the two alleles of the heave chain and one of the four alleles of the kappa and lambda light chains. The constant regions of the heavy chain specify what antibody isotype you are. IgD is membrane-bound. IgM can be membrane-bound or secreted. Abcam (an Ab company)

N C N C N N C C Epitope: the portion of the target antigen (Ag) bound Never before seen epitopes can be recognized Too many epitopes to allow direct encoding in the genome

Fab = Fragment antigen binding Fc = Fragment crystallizable Why name it F(ab’)2? VL CL VH CH1 CH2 CH3 This is how your textbook is going to show it. Each little rectangle is an “Immunoglobulin domain.” Proteases like to cleave antibodies near the “hinge region” because these flexible areas and the most protease-accessible. This flexibility allows an antibody to bind multiple V domains to multivalent antigens with different spacing and orientation. Figure 4.4

Consider the light chain

Ig domain is a recurring protein structure in immunology Immunoglobulin superfamily This is how the textbook will denote: heavy light Figure 4.3

A quick preview of the T cell receptor (TCR). Structurally related to immunoglobulin. Does not recognize free antigen. Instead recognizes peptide antigens in the context of presentation by the major histocompatibility complex (MHC) molecules. Figure 4.13

One difference between Ig and TCR: Ig recognizes Ag in native form whereas TCR recognizes Ag as peptide presented by MHC molecules.

These three regions vary. “They are know as hypervariable regions” or “complementarity-determining regions”. Figure 4.3

Hypervariable regions=CDRs, complementary determining regions AA AA Figure 4.6

Figure 4.7

Another difference between Ig and TCR: TCR solely functions as an antigen-specific receptor, whereas Ig encodes both antigen receptor and the major effector molecule (antibody). Effector functions are mostly mediated by the IgH constant regions.

IgH C regions specify the isotype: IgM, IgD, IgG, IgE, or IgA Antigen (Ag) recognition here Business end of the molecule is here, encoded by heavy chain (IgH) C regions. IgH C regions specify the isotype: IgM, IgD, IgG, IgE, or IgA Now consider the heavy chain

Review. So far… IgG is example 4 polypeptide chains Modular construction Ig superfamily Flexibility in hinge region Heavy chain constant region defines the isotype. Figure 4.1

Your first look at the immunoglobulin genes This is just heavy chain. There is also k and l light chain. Modular construction, just like Ig proteins! Here mainly concerned with the 5’ (V(D)J) part of the locus, and at the end of the lecture (and Thurs) with the 3’ C regions. VH CH1 CH2 CH3 CH4 This is your first look at a gene that encodes immunoglobulin. This is the heavy chain. Lots of detail has been removed. This gizmo is encoded in an ~3 Mb region on mouse chromosome 12q. For obvious evolutionary reasons, a highly similar IgH locus is in a syntenic block on human Ch 14q. V=Variable. D=Diversity. J=Joining. Related to Fig.5.5, but more complex

Your first look at the immunoglobulin genes V=variable (D)=diversity (heavy chain only) J=joining This is just heavy chain. There is also k and l light chain. Modular construction, just like Ig proteins! Here mainly concerned with the 5’ (V(D)J) part of the locus, and at the end of the lecture (and Thurs) with the 3’ C regions. VH CH1 CH2 CH3 CH4 This is your first look at a gene that encodes immunoglobulin. This is the heavy chain. Some detail has been removed. Related to Fig.5.5, but more complex

This is how your Ig loci look while you are a developing embryo, and in every cell of your body except B cells “Gene segments” In any given B cell, either k or l locus is used, not both Heavy chain defines isotype: Cm=IgM Figure 5.5

No need to memorize this chart No need to memorize this chart. Know that there are many V segments, several J segments; D segments restricted to IgH/TCRb Not to scale Figure above only shows protein-coding regions. Regulatory elements in DNA provide control of e.g. recombination Recombination of the TCR loci proceeds through a very similar mechanism. Figure 5.5, 5.4

Figure 4.1

Basically Ig recombination only in the B lineage Figure 5.3 In the heavy chain locus during B cell development (in the bone marrow), D-J recombination occurs first. Basically Ig recombination only in the B lineage Figure 5.3

Figure 4.6, 4.7

CDR3 made up of the junction between gene segments Questions? CDR 1 and 2 encoded in V segment, which vary significantly in this region from segment to segment (positive selection) CDR3 made up of the junction between gene segments Questions? Framework regions are under purifying Darwinian selection, as Ig fold must be preserved. CDRs 1 and 2 are under positive selection, as variability ensures robust B cell responses to a wide variety of antigens. CDR 3 is the product of V(D)J recombination. Figure 5.5, 4.7

Recognized by the RAG-1 and RAG-2 proteins. * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Recombination signal sequences (RSSs, asterisks) mark potential sites of V(D)J recombination. Recognized by the RAG-1 and RAG-2 proteins. The same figure, but with a regulatory feature added: the recombination signal sequence (RSS), immediately downstream of V segments, upstream of J segments, and on both sides of the D segment. Figure 5.5

Recombination signal sequences (RSSs) are recognized by RAG (recombination activating gene) proteins-1 and -2 5’ 5’ 5’ 5’ 12mers/23mers do not recombine with themselves, only with RSSs with the other spacer (the “12/23 rule”) ∴ V segments never recombine with other V segments Figure 5.6

You are looking at an Ig light chain, either k or l… ✔ -or- ✔ and ✔ or… What I showed you before was an over-simplification, as sometimes segments are in either orientation. Figure 5.7

“Direct repeat” RSSs leads to joining and deletion of the intervening DNA “Inverted repeat” RSSs leads to joining and inversion of the intervening sequence. You will have to study the topology of this figure to see it. Figure 5.7

This is the basis of the 12/23 rule Step 1: nick DNA and catalyze inter-strand attack of one strand of the DNA onto the other, using 3’ hydroxyl group of DNA end as nucleophile. This creates the “hairpin ends” in the figure. Figure 5.8

Variability in the cut generates “P-nucleotide varariability” TdT=terminal deoxy-ribonucleotidyl transferase TdT is lymphocyte specific TdT introduces additional nucleotides at the coding junctions, and increasing diversity “N-nucleotide variability” Lack of RAG proteins, or DNA-PK, or ArtemisSCID (C=combined, meaning B and T cells affected) Step 2: nick the hairpin (variation introduced) Step 3: add nucleotides with TdT (variation introduced) Step 4: repair the break using cell type-nonspecific DNA repair proteins such as DNA ligase. Figure 5.8

Figure 5.15 No need to memorize this chart. combinatorial diversity Preview from Chapter 8 that is meaningful here is “allelic exclusion.” It is the reason why a given B (or T) cell makes only one specificity of Ig (or TCR). Many products of these recombination reaction will be duds because they wind up out of frame. These could be fixed by further recombination, or by recombination of the other allele (RAG genes are only switched off if recombination produces a functional gene product). Alternatively, the developing B cells die in the bone marrow. This inefficiency is the price we pay for a diverse repertoire. junctional diversity + somatic hypermutation In B CELLS ONLY (next lecture) Figure 5.15

A difference between Ig and TCR: TCR solely functions as an antigen-specific receptor, whereas Ig encodes both antigen receptor and the major effector molecule (antibody). Effector functions are mostly mediated by the IgH constant regions.

Consequently, the molecular biology of the BCR/antibody (immunoglobulin) gene loci following activation is much more complex

i. e. , gene expression from the Ig loci is increased many fold i.e., gene expression from the Ig loci is increased many fold. In plasma cells (effector B cells), Ig accounts for 50% of all gene expression. This does not happen with T cell receptor. Ig expression is many hundreds of fold higher than the in some ways conceptually analogous TCR in an activated T lymphocyte, because in B cells Ig also encodes the effector molecule.

Conversion to Ab secretion changes cellular moprhology Resting B cell: Antibody secreting (plasma) cell: Atlas of Blood Cells

i.e., there are specific molecular mechanisms that change immunoglobulin from membrane-bound to secreted forms. This does not happen with T cell receptor. Therefore, these processes are unique to B cells…

B cells secrete antibody upon stimulation cell surface B cells become activated by signaling through B cell Ag receptor (BCR) complex, plus costimulatory signals. Same thing at other heavy chain regions. SC: secretion coding; MC: membrane coding Figure 5.22

B cells secrete antibody upon stimulation The switch from membrane-bound to secreted Ig is mediated by REGULATED TRANSCRIPTIONAL TERMINATION to control mRNA SPLICING PATTERNS. cell surface B cells become activated by signaling through B cell Ag receptor (BCR) complex, plus costimulatory signals. Same thing at other heavy chain regions. SC: secretion coding; MC: membrane coding Figure 5.22

i.e., there are further recombination events to generate antibodies with specific effector functions (Ig isotype switching), and site-specific mutagenesis that allows the selection of high-affinity antibodies (affinity maturation) Therefore, these processes are unique to B cells…

If you have not already, read the book chapters before Thursday! I will be relying on figures from your textbook less in the next lecture. Reading the chapters therefore becomes more of an additional rather than redundant resource.